Method for evaluating uniformity of spots in arrays

ABSTRACT

A method whereby the uniformity of spots on a DNA microarray is evaluated by extremely simple means.  
     A method for evaluating a uniformity of spots on a DNA microarray having a plurality of spots, these spots undergoing specific emissions as a result of the hybridization of target DNA and tagged probe DNA, wherein the uniformity of the spots is evaluated by examining whether patterns having periodicity are manifested in a sequence BG comprising background data obtained in a manner described below.  
     &lt;Method for Preparing the Sequence BG&gt; 
     (1) By applying an analysis software to images obtained by scanning the monochromatic emission of the DNA microarray, background data for each spot are obtained.  
     (2) Concerning each spot, the corresponding plate No.(α) of the target DNA and position (β, Γ) are determined, wherein a is a symbol or a numerical symbol identifying the plate while β and Γ respectively stand for the row and column of a matrix formed by plate holes.  
     (3) The No.(α) and the position (β, Γ) on the plate are assigned to respective background data.  
     (4) The sequence BG consisting of the background data aligned in the orders of the plate No.(α) (the first priority) and the position (β, Γ) on the plate(the second priority) is obtained.

FIELD OF THE INVENTION

[0001] The present invention relates to a method for evaluating theuniformity of spots printed on an array, and is a technique supportingthe analysis of an array having spots located two-dimensionally on asubstrate, such as a DNA microarray, a DNA chip, a protein array, etc.

BACKGROUND OF THE INVENTION

[0002] Research involving the analysis of gene information, as typifiedby the Human Genome Project, is occurring at an ever faster paceworldwide, bringing with it an increasing need for new methodologiescapable of efficiently analyzing expressions at the in-vivo gene level.

[0003] A new method for measuring gene expression levels in cells is aDNA microarray, wherein several hundred to several tens of thousands ofsamples of DNA are aligned and fixed in spots in a matrix shape to aglass slide. mRNA (the target) that has been extracted and purified fromtarget cells is hybridized on the DNA microarray.

[0004] Fundamentally, the common method for performing measurementsusing DNA microarrays is two-color fluorescence labeling. In thismethod, mRNA originating from two types of cells (for example, normalcells and cancer cells) is extracted and purified, and the cells arelabeled with fluorescent materials (CY3 and CY5) that have mutuallydiffering excitation wave lengths. Then, competitive hybridization isperformed on the same spots on the DNA microarray, and the fluorescentintensity of each spot on the array is measured by using two channels(CH1 and CH2) to view the mutually differing excitation wave lengths CY3and CY5. By this means, the comparative quantity of gene levelexpression of the two types of cells is measured. In practice, after thefluorescent signals have been measured, the measurement results aresuperposed and color analysis is performed.

[0005] The data from CY3 and CY5 is normally calculated based on thefollowing relational expression, with essentially the value of log (R/G)being utilized as the gene expression data.

[0006] (Sample A CY3 fluorescence data)

[0007] CH1-CH1B (Channel 1 background)=data R of CY3 (red)

[0008] (Sample B CY5 fluorescence data)

[0009] CH2-CH2B (Channel 2 background)=data G of CY5 (green)

[0010] Here, CH1 (channel 1) and CH2 (channel 2) are the measuredfluorescent intensity values of the spots (channel 1 and 2 existing soas to measure red and green separately), measured using a laser scanner.Further, CH1B (background data of channel 1) and CH2B (background dataof channel 2) are background data of the spots measured using a laserscanner.

[0011] Gene spots with a greater degree of gene expression in Sample Ashow as red, spots with a greater degree of gene expression in Sample Bshow as green, and spots with an approximately equal degree of geneexpression show as yellow. That is, the spots show the following colors,in accordance with the ratio of R to G:

[0012] R/G>1 Red

[0013] R/G=1 Yellow

[0014] R/G<1 Green

[0015] As research based on DNA microarray data, such as the analysis ofperiodicity between genes, gene expression networks, and gene transfercontrol cascades, is being developed, and mathematical informationalmethods of this second generation research crucially require improvedaccuracy. A high degree of reliability is required with respect to thedata from DNA microarrays.

[0016] Further, highly accurate data is required in the case wherecancer is diagnosed on the basis of gene expression data.

[0017] However, gene analysis using DNA microarrays has only recentlybegun, and there are many cases where the issue of reproducibility needsto be resolved.

[0018] In particular, it is known that printing, hybridization,processing of the slide surface, etc. readily causes changes in theshape and size of the spots, and that mechanical influences during thespotting process, such as the minute displacement, vibration, etc. ofthe printing pins or platform, readily cause changes in the position ofthe spots.

[0019] Although the uniformity of the spots on the DNA microarray is animportant factor that affects the accuracy of signal data, a method forevaluating this uniformity does not exist.

[0020] The present invention presents an extremely simple method forevaluating the uniformity of the spots on an array such as a DNAmicroarray, etc.

DISCLOSURE OF THE INVENTION

[0021] Portions of the gene expression data from DNA microarrays havebeen made public for researchers to use. This gene expression data havebeen made public by Stanford University, MIT, and Harvard University.Stanford University have DNA microarray data base. In 1997, ProfessorBrown's group at Stanford University succeeded in analyzing, for thefirst time in the world, the total gene expression (6400 genes) of theyeast cell (this is also public data).

[0022] Inventors, who were concerned that there was some regularity inthe gene expression quantities, obtained the total gene expression dataof the yeast cell (TUP1) that was made public in the DNA microarray database of Stanford University, rearranged the expression data in thechromosome order and gene order, and analyzed the gene expression datathus obtained.

[0023] The result was that, on multiplying by 2, the presence of weakperiodicity was ascertained. However, these periods did not originatefrom the gene expression levels, but appeared due to the influence ofbackground data.

[0024] The present inventors have discovered a relationship between thepresence of periodicity in the background data (this background databeing used as fluorescence intensity compensatory data) and thenon-uniformity of spots, and have discovered that the presence ofperiodicity in the background data furthermore exerts an importantinfluence on the analysis of gene expression data. The present inventionhas resulted from the further discovery by the inventors that thisinformation can be applied not only to DNA microarrays, but also to allarrays having spots located two-dimensionally on a substrate, such asDNA chips, protein arrays, etc.

[0025] That is, the present invention is a method for evaluating auniformity of spots on an array having a plurality of spots, these spotsundergoing specific emissions as a result of the hybridization of targetmatter and tagged probe matter, wherein the uniformity of the spots isevaluated by examining whether patterns having periodicity aremanifested in a sequence BG comprising background data obtained in amanner described below.

[0026] <Method for Preparing the Sequence BG>

[0027] (1) By applying an analysis software to images obtained byscanning the monochromatic emission of the array, background data foreach spot are obtained.

[0028] (2) Concerning each spot, the corresponding plate No.(α) of thetarget matter and position (β, Γ) are determined, wherein α is a symbolor a numerical symbol identifying the plate while β and Γ respectivelystand for the row and column of a matrix formed by plate holes.

[0029] (3) The No.(α) and the position (β, Γ) on the plate are assignedto respective background data.

[0030] (4) The sequence BG consisting of the background data aligned inthe orders of the plate NO.(α) (the first priority) and the position (β,Γ) on the plate(the second priority) is obtained.

[0031] According to the present invention, the uniformity of spots on anarray such as a DNA microarray or the like can be evaluated in anextremely simple manner.

[0032] According to the method of the present invention, the signal dataobtained from an array such as a DNA microarray, or the like, can beanalyzed with great accuracy by evaluating the uniformity of the spotson the array in advance.

BRIEF DESCRIPTION OF THE DRAWINGS

[0033]FIG. 1 shows a flow chart displaying the commonly used steps foranalyzing signal data using a DNA microarray.

[0034]FIG. 2 shows a commonly seen distribution of one spot within anallocated square.

[0035]FIG. 3 shows an example of sequence I_(j) (j=1, . . . , 100)displayed in a matrix group.

[0036]FIG. 4 shows an example of background data (CH2B) of yeast cellDNA microarray data displayed as a colored matrix, the relationshipbetween CH2B and the colors being shown in the lower row.

[0037]FIG. 5 shows an example of background data (CH1B) of the yeastcell DNA microarray data displayed as a colored matrix, the relationshipbetween CH1B and the colors being shown in the lower row.

[0038]FIG. 6 shows signal data (CH1D) of channel 1 in a DNA microarrayimage wherein color is converted in accordance with the size of numbervalues (Embodiment 1).

[0039]FIG. 7 shows signal data (CH2D) of channel 2 in a DNA microarrayimage wherein color is converted in accordance with the size of numbervalues (Embodiment 1).

[0040]FIG. 8 shows an example of background data (CH1B) of melanoma DNAdisplayed as a colored matrix, the relationship between CH1B and thecolors being shown in the lower row.

[0041]FIG. 9 shows an example of background data (CH2B) of the melanomaDNA displayed as a colored matrix, the relationship between CH2B and thecolors being shown in the lower row.

[0042]FIG. 10 shows signal data (CH1D) of channel 1 in a DNA microarrayimage wherein color is converted in accordance with the size of numbervalues (Embodiment 2).

[0043]FIG. 11 shows signal data (CH2D) of channel 2 in a DNA microarrayimage wherein color is converted in accordance with the size of numbervalues (Embodiment 2).

[0044]FIG. 12 shows an outline of the yeast cell DNA microarray data.

PREFERRED ASPECT TO EMBODY THE INVENTION

[0045] Below, a description is given using a DNA microarray as anexample. A flow chart displaying the commonly used steps for analyzingsignal data using a DNA microarray is as shown in FIG. 1.

[0046] <Background Data>

[0047] When scanning is performed on a slide on which hybridization hasbeen completed, the color status of each spot on the slide is recordedas picture image data. Then, the picture image data is processed usinganalysis software, and color data of the spots is acquired.Specifically, the image obtained by scanning is overlaid with a gridimage having a plurality of squares, one square being allocated for eachspot, and then the signal data and background data within each squareare acquired (FIG. 2).

[0048] Usually, the signal data is measured as the color intensitywithin an oval-shaped spot wherein a major axis and minor axis have beendesignated. The background data is measured as the intensity within thesquare surrounding the spot and in the area outside the boundaries ofthe spot.

[0049] The background data should be measured when all the spots arevisible and emit at a brightness whereby the emission intensity is notsaturated. There can be a slight variation in the background datadepending on the detecting conditions of the emission signal of thescanning device or the method used by the analysis software for samplingthe spots. However, the method of the present invention is not affectedby the scanning device or the type of analysis software.

[0050] Preferred scanning devices are GenePix 4000A, GeneTACLSIV,GTMASS, GMS418Array Scanner, AvalancheMicroscanner, ChipReader,GeneTAC2000, CRBIO, ScanArray3000, 4000, 5000, etc.

[0051] Preferred analysis software is ScanAlyze, ArrayAnalyzer, ImaGene,AutoGene, QuantArray, QuantarrayAutomation, MicroArraySuite,ArrayVision, ArrayGauge, GenePixPro, etc.

[0052] <Method for Preparing Sequence BG>

[0053] In the present invention, a sequence BG serving as an indicatorfor revealing the characteristics of the background data is prepared asfollows.

[0054] (1) By applying an analysis software to images obtained byscanning the monochromatic emission of the DNA microarray, backgrounddata for each spot are obtained.

[0055] (2) Concerning each spot, the corresponding plate No.(α) of thetarget DNA and position (β, Γ) are determined, wherein a is a symbol ora numerical symbol identifying the plate while β and Γ respectivelystand for the row and column of a matrix formed by plate holes.

[0056] (3) The No.(α) and the position (β, Γ) on the plate are assignedto respective background data.

[0057] (4) The sequence BG consisting of the background data aligned inthe orders of the plate NO.(α) (the first priority) and the position (β,Γ) on the plate(the second priority) is obtained.

[0058] <Display Methods for Patterns in the Sequence BG>

[0059] There is no particular restriction on the display methods of thepresent invention as long as these can be used to determine the presencein the sequence BG of patterns having periodicity.

[0060] Preferred display methods are the methods below.

[0061] (Display Method 1)

[0062] A method whereby a sub-sequence formed from 1 or more elements isextracted from a sequence, each number contained in the sub-sequenceforming a color dot, wherein hue, luminosity, saturation, or acombination thereof, is defined by each type of number, the color dotfurther being sequentially output in a color dot matrix arranged in amatrix shape, a color pattern obtained from the output of the color dotmatrix causing the intrinsic regularity to be revealed.

[0063] (Display Method 2)

[0064] A method whereby a sequence is divided into a plurality ofsub-sequences, each number contained in the divided sub-sequencesforming a sub-color dot column wherein hue, luminosity, saturation, or acombination thereof, is defined by each type of number, the sub-colordot columns being arranged in an aligned manner to output a color dotmatrix wherein the color dots are arranged in a matrix shape, a colorpattern obtained from the output of the color dot matrix causing latentcharacteristics within the sequence to be revealed.

[0065] A more preferred method is display method 3 below.

[0066] (Display Method 3)

[0067] A method whereby, in display method 1 or display method 2, eachnumber forming a sequence I_(j) (j=1, . . . , m) is arranged accordingto the following positioning pattern: (j = 1, 2, 3, …  , k)(j = k + 1, k + 2, k + 3, …  , k + k)⋮(j = (n − 1)k + 1, (n − 1)k + 2, (n − 1)k + 3, …  , (n − 1)k + k)(j = nk + 1, nk + 2, nk + 3, …  , nk + k)

[0068] (here, k is an integer of 2 or more, n is a natural number suchthat nk+1≦m≦nk+k), a color dot matrix is output, latent characteristicswithin the sequence being revealed.

[0069] An even more preferred method is display method 4 below.

[0070] (Display Method 4)

[0071] A method whereby, when p is any given natural number less than m,and r is any given natural number, when the display method of displaymethod 3 is implemented while substituting k=p, p+r, p+2r, p+3r, . . . ,a color dot matrix group is output wherein a color dot matrix of the pcolumn, a color dot matrix of the p+r column, and color dot matrices ofthe p+2r, p+3r . . . columns, as below, are all arranged in an alignedmanner, latent characteristics within the sequence being revealed.

[0072] Display method 4 is particularly effective in the case whererepeated units are totally unclear, or in the case where a portion of asequence simply does not exist in repeated regions.

[0073]FIG. 3 schematically shows a method for arranging each element insequence I_(j) (j=1 , . . . , 100) obtained by means of method 4 asmatrix groups consisting of the matrices k=1, k=2, k=3, k=4, . . . andk=20.

[0074] <Method for Evaluating Uniformity of Spots>

[0075] When the sequence BG has been displayed by means of a suitabledisplay method, it is verified whether patterns having periodicity arepresent. In the case where patterns having periodicity are present, itcan be determined that the spots on the DNA microarray have lowuniformity.

[0076] Various types of patterns having periodicity can be present inthe sequence BG, such as a constantly repeated pattern or a plurality oftypes of patterns repeated across the entire sequence BG, patterns beingrepeated in portions of the sequence BG, etc.

[0077] If patterns having periodicity are not present in the sequenceBG, it can be determined that the spots were printed uniformly, andconsequently highly accurate data analysis is possible. On the otherhand, if patterns having periodicity are present in even a portion ofthe sequence BG, the printing conditions of the spots were not uniform.Consequently the signal data has low reliability, and is influenced bycompensatory factors, namely the patterns having periodicity of thebackground data. Since the periodic noise is included for signal data,the precise analysis is difficult.

[0078] Below, the method of the present invention is described moreconcretely.

[0079] <Embodiment 1>

[0080] (Yeast Cell DNA Microarray)

[0081] The yeast cell DNA microarray data from the DNA microarray datapublished by Stanford University was obtained (Genomic ExpressionPrograms in the Response of Yeast Cells to Environmental Changes, ArrayData File: y11n121 (variable heat 21C)).

[0082] An outline of the data that was obtained is shown in FIG. 12. InFIG. 12, ‘CH1B’ is background data of channel 1, and ‘CH2B’ isbackground data of channel 2 . ‘CH1D’ and ‘CH2D’ are signal data ofcompensated spots obtained according to the following formula.

CH 1 I−CH 1 B=CH 1 D

CH 2 I−CH 2 B=CH 2 D

[0083] Further, in FIG. 12, ‘PLAT’ is a symbol or number identifyingplates, ‘PROW’ is a symbol showing the rows of each plate, and ‘PCOL’ isa number showing the columns of each plate.

[0084] In FIG. 12, data concerning specific target DNA is displayed inrow units. A plate number (‘PLAT’) and position on the plate (‘PROW’,‘PCOL’) is assigned for the background data ‘CH1B’ and ‘CH2B’ for eachitem of target DNA displayed in the ‘NAME’ column. Each item of data islisted first in the plate number unit, then in the plate row unit, andfinally in the plate column unit. Consequently, the order from top tobottom in FIG. 12 forms an order based on method 4 of the sequence BG ofthe present invention.

[0085] First, the background data (CH1B) is listed in sequence in thecolumn ‘CH1B’ of FIG. 12 from the top line to the bottom line to formthe sequence BG, this being displayed according to display method 4.Consequently, the presence of 384 repeat structures in the sequence BGis shown. This result is shown in FIG. 5, wherein each number in thesequence BG is displayed in a matrix shape according to display method 3(here, k=384).

[0086] Similarly, FIG. 4 shows the result wherein each number in thesequence BG of the background data (CH2B) is displayed in a matrix shapeaccording to display method 3.

[0087] It can be seen from these figures that, in both the sequences BGof the background data CH1B and CH2B, periodically fluctuating patternshaving distinct periodicity are present as 384 repeat units.

[0088]FIG. 6 shows the signal data ‘CH1D’ of channel 1 in a DNAmicroarray image wherein color is converted in accordance with the sizeof number values. Further, FIG. 7 shows a DNA microarray image of thesignal data ‘CH2D’ of channel 2 processed in the same way. These DNAmicroarray images are not necessarily identical to actual scannedimages, but schematically show the emission intensity of the spots inscanned images. The non-uniformity of the spots cannot be recognized atall from FIGS. 6 and 7, but since the distinct repeatability in thebackground data can be recognized from FIGS. 4 and 5, the non-uniformityof the spots can be determined.

[0089] Since the background data are utilized as compensatory numbervalues of the fluorescent intensity, if there is periodicity in thebackground data itself, periodicity originating from the background datawill necessarily be present in the gene expression data of the DNAmicroarray. Since this type of periodicity in the background dataaffects the reliability of the DNA microarray data, care is requiredduring data analysis.

[0090] <Embodiment 2>

[0091] (Melanoma DNA Microarray)

[0092] The melanoma DNA microarray data from the DNA microarray datapublished by Stanford University was obtained (NC160 Cancer MicroarrayProject).

[0093] As with embodiment 1, background data (CH1B) is prepared, and isdisplayed according to display method 4. Consequently, the presence of96 repeat structures in a sequence BG is shown (although it has alsobeen suggested that 24 repeat structures are intrinsic to the sequenceBG). FIG. 8 shows this result, wherein each number in the sequence BG isdisplayed in a matrix shape according to display method 3 (here, k=96).

[0094] Similarly, FIG. 9 shows this result, wherein each number in thesequence BG of the background data (CH2B) is displayed in a matrix shapeaccording to display method 3.

[0095] In these figures, a plurality of vertical lines (that is, repeatsof 96 units) are displayed distinctly, and it can be understood thatpatterns having periodicity are present in the sequences BG of thebackground data of CH1B and CH2B.

[0096] As with embodiment 1, the signal data ‘CH1D’ of channel 1 and thesignal data ‘CH2D’ of channel 2 are shown in DNA microarray images inFIGS. 10 and 11. The non-uniformity of spots therein cannot berecognized at all from FIGS. 10 and 11, but since the distinctrepeatability in the background data can be recognized from FIGS. 8 and9, the non-uniformity of the spots can be determined.

[0097] According to the present invention, the uniformity of the spotson an array such as a DNA microarray or the like can be evaluated in anextremely simple manner.

[0098] According to the method of the present invention, by evaluatingin advance the uniformity of the spots on the array, the signal dataobtained from an array such as a DNA microarray or the like can beanalyzed with great accuracy.

[0099] Specific examples of embodiments of the present invention arepresented above, but these merely illustrate some possibilities of theinvention and do not restrict the claims thereof. The art set forth inthe claims includes transformations and modifications to the specificexamples set forth above.

[0100] Furthermore, the technical elements disclosed in the presentspecification or figures may be utilized separately or in all types ofconjunctions and are not limited to the conjunctions set forth in theclaims at the time of submission of the application. Furthermore, theart disclosed in the present specification or figures may be utilized tosimultaneously realize a plurality of aims or to realize one of theseaims.

1. A method for evaluating a uniformity of spots on an array having aplurality of spots, these spots undergoing specific emissions as aresult of the hybridization of target matter and tagged probe matter,wherein the uniformity of the spots is evaluated by examining whetherpatterns having periodicity are manifested in a sequence BG comprisingbackground data obtained in a manner described below. <Method forPreparing the Sequence BG> (1) By applying an analysis software toimages obtained by scanning the monochromatic emission of the array,background data for each spot are obtained. (2) Concerning each spot,the corresponding plate No.(α) of the target matter and position (β, Γ)are determined, wherein α is a symbol or a numerical symbol identifyingthe plate while β and Γ respectively stand for the row and column of amatrix formed by plate holes. (3) The No.(α) and the position (β, Γ) onthe plate are assigned to respective background data. (4) The sequenceBG consisting of the background data aligned in the orders of the plateNO.(α) (the first priority) and the position (β, Γ) on the plate(thesecond priority) is obtained.
 2. A method for evaluating a uniformity ofspots on a DNA microarray having a plurality of spots, these spotsundergoing specific emissions as a result of the hybridization of targetDNA and tagged probe DNA, wherein the uniformity of the spots isevaluated by examining whether patterns having periodicity aremanifested in a sequence BG comprising background data obtained in amanner described below. <Method for Preparing the Sequence BG> (1) Byapplying an analysis software to images obtained by scanning themonochromatic emission of the DNA microarray, background data for eachspot are obtained. (2) Concerning each spot, the corresponding plateNo.(α) of the target DNA and position (β, Γ) are determined, wherein ais a symbol or a numerical symbol identifying the plate while β and Γrespectively stand for the row and column of a matrix formed by plateholes. (3) The No.(α) and the position (β, Γ) on the plate are assignedto respective background data. (4) The sequence BG consisting of thebackground data aligned in the orders of the plate NO.(α) (the firstpriority) and the position (β, Γ) on the plate(the second priority) isobtained.
 3. A method as set forth in claim 1, the method beingcharacterized in that the patterns in the sequence BG are displayedaccording to display method 1 or display method 2 described below,whereby it is determined whether the patterns having periodicity aremanifested in the sequence BG. <Display Methods> (Method 1) A methodwhereby a sub-sequence formed from 1 or more elements is extracted froma sequence, each number contained in the sub-sequence forming a colordot, wherein hue, luminosity, saturation, or a combination thereof, isdefined by each type of number, the color dot further being sequentiallyoutput in a color dot matrix arranged in a matrix shape, a color patternobtained from the output of the color dot matrix causing the intrinsicregularity to be revealed. (Method 2) A method whereby a sequence isdivided into a plurality of sub-sequences, each number contained in thedivided sub-sequences forming a sub-color dot column wherein hue,luminosity, saturation, or a combination thereof, is defined by eachtype of number, the sub-color dot columns being arranged in an alignedmanner to output a color dot matrix wherein the color dots are arrangedin a matrix shape, a color pattern obtained from the output of the colordot matrix causing latent characteristics within the sequence to berevealed.
 4. A method as set forth in claim 3, the method beingcharacterized in that, in display method 1 or display method 2, eachnumber forming a sequence I_(j) (j=1, . . . , m) is arranged accordingto the following positioning pattern: (j = 1, 2, 3, …  , k)(j = k + 1, k + 2, k + 3, …  , k + k)  ⋮(j = (n − 1)k + 1, (n − 1)k + 2, (n − 1)k + 3, …  , (n − 1)k + k)(j = nk + 1, nk + 2, nk + 3, …  , nk + k)

(here, k is an integer of 2 or more, n is a natural number such thatnk+1≦m≦nk+k), a color dot matrix is output, latent characteristicswithin the sequence being revealed.
 5. A method as set forth in claim 4,the method being characterized in that when p is any given naturalnumber less than m, and r is any given natural number, when the displaymethod set forth above is implemented while substituting k=p, p+r, p+2r,p+3r, . . . , a color dot matrix group is output wherein color dotmatrices of p, p+r, p+2r, p+3r . . . are all arranged in an alignedmanner.
 6. A method as set forth in claim 2, the method beingcharacterized in that the patterns in the sequence BG are displayedaccording to display method 1 or display method 2 described below,whereby it is determined whether the patterns having periodicity aremanifested in the sequence BG. <Display Methods> (Method 1) A methodwhereby a sub-sequence formed from 1 or more elements is extracted froma sequence, each number contained in the sub-sequence forming a colordot, wherein hue, luminosity, saturation, or a combination thereof, isdefined by each type of number, the color dot further being sequentiallyoutput in a color dot matrix arranged in a matrix shape, a color patternobtained from the output of the color dot matrix causing the intrinsicregularity to be revealed. (Method 2) A method whereby a sequence isdivided into a plurality of sub-sequences, each number contained in thedivided sub-sequences forming a sub-color dot column wherein hue,luminosity, saturation, or a combination thereof, is defined by eachtype of number, the sub-color dot columns being arranged in an alignedmanner to output a color dot matrix wherein the color dots are arrangedin a matrix shape, a color pattern obtained from the output of the colordot matrix causing latent characteristics within the sequence to berevealed.